1. Field of the Invention
The present invention relates to reagents and methods to validate the results obtained in a nucleic acid testing assay. More particularly, the present invention relates to biological reagents and methods to verify the efficiency of sample preparation and nucleic acid amplification and/or detection.
2. Description of the Related Art
With the advent of molecular biology, nucleic acid testing (NAT) assays are becoming increasingly popular. These assays rely on the hybridization of synthetic oligonucleotide primers and/or probes targeting a nucleotide sequence of the organism(s) of interest. Highly sensitive NAT technologies, such as the widely used polymerase chain reaction (PCR), represent important tools in the field of molecular diagnostics. Since the discovery of PCR in 1983, numerous DNA-based assays targeting a wide variety of microbial pathogens have been developed (Nolte and Caliendo, 2003, Molecular detection and identification of microorganisms, p. 234-256, In Manual of Clinical Microbiology (8th ed.), Murray et al., American Society for Microbiology, Washington, D.C.; Lee et al. 1997, Nucleic Acid Amplification Technologies: Application to Disease Diagnosis, Biotechniques Books, Eaton Publishing, Boston, Mass.; Persing et al., 1993, Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.; Ehrlich; Greenberg, 1994, PCR-based Diagnostics in Infectious Disease, Blackwell Scientific Publications, Boston, Mass.). Many of these NAT assays have been designed for microbial detection directly from clinical, environmental or food samples, which are known to contain inhibitors of NAT assays. Over the years, a variety of procedures performed from a variety of test samples for concentration and/or purification of cells or viral particles as well as for release, concentration and/or purification of nucleic acids have been developed. However, samples prepared by these procedures may still contain impurities that interfere with NAT assays.
Numerous studies have demonstrated that many types of clinical, environmental and food specimens may contain substances interfering with nucleic acid amplification processes including PCR, ligase chain reaction (LCR), transcription-mediated amplification (TMA) and strand displacement amplification (SDA) (Courtney et al. 1999, Analytical Biochem. 270:249-256; Rosenstraus et. al., 1998, J. Clin. Microbiol. 36: 191-197; Morre et al. 1996, J. Clin. Microbiol. 34:3108-3114; Lee et al. 1997, Nucleic Acid Amplification Technologies: Application to Disease Diagnosis, Biotechniques, Books, Eaton Publishing, Boston, Mass.; Persing et al., 1993, Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.; Ehrlich and Greenberg, 1994, PCR-based Diagnostics in Infectious Disease, Blackwell Scientific Publications, Boston, Mass.). Therefore, it is crucial to identify inhibitory test samples because negative test results may be attributable to inhibition of the NAT assay by impurities not eliminated, neutralized or inactivated by the protocol used for sample preparation. Inhibitory test samples can be identified by verifying the efficiency of amplification and/or detection of a nucleic acid target serving as a control. Such control may be external when the control nucleic acid is added to a portion of the sample tested in parallel for amplification and/or detection of the control target sequences while another portion of the sample is analyzed for amplification and/or detection of the analyte target sequences to be detected by the assay. Such control is called an “internal control (IC)” when both control target sequences and analyte target sequences are purified and/or detected in the same reaction vessel. More specifically, this internal control system provides an IC for amplification and/or detection (ICAD) of nucleic acids. Inhibitory samples lead to lower or no ICAD signal while a positive signal for the ICAD of the expected intensity demonstrates the absence of nucleic acid amplification/detection inhibitors in the test sample, thereby validating a negative result for the primary target(s). ICAD have been developed by using various strategies (Courtney et al., 1999, Analytical Biochem., 270:249-256; Rosenstraus et al. 1998, J. Clin. Microbiol., 36:191-197; Morre et al., 1996, J. Clin. Microbiol., 34:3108-3114; Stocher et al., 2002, J. Virol. Methods 108:1-8). The IC target nucleic acids may be cloned in diverse cloning vectors including plasmids, cosmids, bacteriophages and transposons (Sambrook and Russel, 2001, Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
It is also important to validate the sample preparation method for NAT assays to ensure its efficiency to lyse microbial cells because many microbial species have a thick and/or sturdy cell wall (e.g. gram-positive bacteria, mycobacteria, bacterial spores and yeasts), which make them difficult to lyse. Efficient cell lysis is required to release target nucleic acids to allow their amplification and/or detection. Furthermore, the current trend in molecular diagnostics is to integrate sample preparation and nucleic acid amplification and/or detection into a single device. Therefore, there is a need to develop strategies to validate both the sample preparation procedure and the amplification and/or detection processes.
The inventors have previously demonstrated that popular commercially available kits for rapid microbial DNA isolation were not efficient for nucleic acid recovery from gram-positive bacteria and yeast cells (PCT patent publication WO 03/008636). U.S. Pat. Nos. 5,994,078 and 6,074,825 describe a method to prepare stable encapsulated reference nucleic acids used to monitor genetic testing by providing an external control reaction to verify the efficiency of sample preparation for NAT assays. However, obtaining such sample preparation control vehicles requires chemical and/or physical treatment of the cellular vehicle used to mimic the membrane stability of the test cells. Consequently, the modified cellular vehicle is different from the naturally encountered microbial cells targeted by NAT assays. Moreover, such external controls do not allow a monitoring of the efficiency of amplification and/or detection procedures.
There thus remains a need to provide a means to verify the efficiency of a sample preparation procedure and of the performance of nucleic acid amplification and/or detection. There also remains a need to provide biological reagents to enable such methods. In addition there remains a need to provide methods and reagents, which more truly validate the results obtained with a NAT assay.
The present invention seeks to meet these and other means.